Pathogens have evolved a number of ways to subvert host defense arsenals to persist long term and induce recurrent infections. We have previously shown that uropathogenic E. coli (UPEC) persists as quiescent reservoirs in membrane-bound compartments within the bladder wall and causes frequent and recurrent urinary tract infections (UTIs). How these reservoirs form and persist is not known; this lack of knowledge impedes our ability to develop treatments to prevent recurrent UTIs. One mechanism used by animals to control infection by intracellular pathogens is autophagy, an evolutionarily conserved process that is activated under starvation or stress to recycle nutrients and damaged membranes by delivering them to the lysosome for degradation. We recently showed that an autophagy gene, Atg16L1, plays a key role in UTI pathogenesis: a mutation in the Atg16L1 gene limits the ability of UPEC to persist and cause recurrent UTIs and is associated with urothelial architectural defects. These defects lead to alterations in complex membrane recycling events in superficial urothelial cells required for both the normal function of the bladder and UPEC pathogenesis. The objective of this application is to determine the mechanisms by which UPEC hijack the normal vesicle trafficking of the bladder epithelial cells to form persistent reservoirs. The centrl hypothesis tested is that UPEC avoid destruction by lysosomes in urothelial cells by appropriating the autophagy pathway, and that Atg16L1 deficiency re-routes bacteria to an intracellular compartment where they cannot persist. We propose to test this hypothesis as follows: in Aim 1, we will systematically disrupt urothelial cell architecture by using newly established urothelial-specific cre mice driving loss of function of Atg16L1 and other autophagy pathway proteins to determine both the nature of the intracellular UPEC niche in vivo and the role of autophagy in UPEC persistence. In Aim 2, we will use a urothelial cell culture model to elucidate how modulation of Atg16L1 alters the process of UPEC invasion, intracellular survival into urothelial cells, and egress out of cells. We anticipate that our work will provide new insighs into the genetic and molecular interplay between the autophagy pathway and UPEC during a UTI and will provide cellular targets for development of therapeutic interventions to combat recurrent infections. Given that urinary tract infections are common and costly and that antibiotic-resistant pathogens are becoming increasingly prevalent, the potential of this knowledge to contribute to development of new treatment regimens to limit or eradicate sources of recurrent UTI could be vital.